Kinetic characterization of l-[(3)H]glutamate uptake inhibition and increase oxidative damage induced by glutaric acid in striatal synaptosomes of rats.
Glutaric acidemia type I (GA-I) is an inherited metabolic disease characterized by accumulation of glutaric acid (GA) and striatal degeneration. Although growing evidence suggests that excitotoxicity and oxidative stress play central role in the neuropathogenesis of this disease, mechanism underlying striatal damage in this disorder is not well established. Thus, we decided to investigate the in vitro effects of GA 10nM (a low concentration that can be present initial development this disorder) on l-[(3)H]glutamate uptake and reactive oxygen species (ROS) generation in synaptosomes from striatum of rats. GA reduced l-[(3)H]glutamate uptake in synaptosomes from 1 up to 30min after its addition. Furthermore, we also provided some evidence that GA competes with the glutamate transporter inhibitor l-trans-pyrrolidine-2,4-dicarboxylate (PDC), suggesting a possible interaction of GA with glutamate transporters on synaptosomes. Moreover, GA produced a significant decrease in the V(MAX) of l-[(3)H]glutamate uptake, but did not affect the K(D) value. Although the GA did not show oxidant activity per se, it increased the ROS generation in striatal synaptosomes. To evaluate the involvement of reactive species generation in the GA-induced l-[(3)H]glutamate uptake inhibition, trolox (0.3, 0.6 and 6muM) was added on the incubation medium. Statistical analysis showed that trolox did not decrease inhibition of GA-induced l-[(3)H]glutamate uptake, but decreased GA-induced reactive species formation in striatal synaptosomes (1, 3, 5, 10, 15 and 30min), suggesting that ROS generation appears to occur secondarily to glutamatergic overstimulation in this model of organic acidemia. Since GA induced DCFH oxidation increase, we evaluate the involvement of glutamate receptor antagonists in oxidative stress, showing that CNQX, but not MK-801, decreased the DCFH oxidation increase in striatal synaptosomes. Furthermore, the results presented in this report suggest that excitotoxicity elicited by low concentration of GA, could be in part by maintaining this excitatory neurotransmitter in the synaptic cleft by non-competitive inhibition of glutamate uptake. Thus the present data may explain, at least partly, initial striatal damage at birth, as evidenced by acute bilateral destruction of caudate and putamen observed in children with GA-I.
Magni
Danieli Valnes
DV
Centro de Ciências da Saúde, Laboratório de Psicofarmacologia e Neurotoxicidade, Departamento de Fisiologia, Universidade Federal de Santa Maria, 97105-900 Santa Maria, RS, Brazil.
Furian
Ana Flávia
AF
Oliveira
Mauro Schneider
MS
Souza
Mauren Assis
MA
Lunardi
Fabiane
F
Ferreira
Juliano
J
Mello
Carlos Fernando
CF
Royes
Luiz Fernando Freire
LF
Fighera
Michele Rechia
MR
D027322:Amino Acid Transport System X-AG / Q000187:drug effects / Q000378:metabolism; D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D001667:Binding, Competitive / Q000187:drug effects / Q000502:physiology; D002839:Chromans / Q000494:pharmacology; D003342:Corpus Striatum / Q000187:drug effects / Q000378:metabolism* / Q000503:physiopathology*; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D018691:Excitatory Amino Acid Antagonists / Q000494:pharmacology; D018698:Glutamic Acid / Q000378:metabolism*; D005977:Glutarates / Q000378:metabolism* / Q000633:toxicity; D008297:Male; D054069:Multiple Acyl Coenzyme A Dehydrogenase Deficiency / Q000378:metabolism / Q000503:physiopathology; D018384:Oxidative Stress / Q000187:drug effects / Q000502:physiology*; D017729:Presynaptic Terminals / Q000187:drug effects / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D017382:Reactive Oxygen Species / Q000378:metabolism; D009435:Synaptic Transmission / Q000187:drug effects / Q000502:physiology; D013574:Synaptosomes
D027322:Amino Acid Transport System X-AG; D000975:Antioxidants; D002839:Chromans; D004791:Enzyme Inhibitors; D018691:Excitatory Amino Acid Antagonists; D005977:Glutarates; D017382:Reactive Oxygen Species; D018698:Glutamic Acid; C035736:glutaric acid; C010643:6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid